This invention is related to the field of cancer diagnostics and therapeutics.
p53 is the most commonly inactivated gene in human cancer and loss of critical p53 pathways are central to tumorigenesis1,2. The p53 protein binds specific DNA sequences and transcriptionally activates responsive genes. p53 gene mutations that occur in human cancer produce abnormal p53 proteins that are unable to bind DNA and promote the transcription and modulation of its target genes3. Furthermore, mutant p53 protein can act in a dominant negative manner by disturbing the function of wild type p53 protein and its ability to regulate cell proliferation4,5. p53 mutant protein also heterodimerizes with wild type p53 and results in a conformational change of the protein that no longer binds to p53 regulating cis-elements6,7.
There is a continuing need in the art for a more complete understanding of the components of the pathways in which p53 acts. Moreover, there is a continuing need for improved diagnostic and therapeutic methods for treating cancers.
It is an object of the present invention to provide an isolated and purified protein useful for diagnosis and classification of cancers.
It is another object of the present invention to provide a fusion protein useful for raising antibodies and drug screening.
It is an object of the present invention to provide a preparation of antibodies useful for therapeutic intervention in cancer.
Another object of the invention is to provide a cDNA molecule, a subgenomic polynucleotide, and a nucleic acid construct which encode a protein useful for diagnosis and classification of cancers.
Another object of the invention is to provide methods of diagnosing and classifying neoplastic tissues of humans.
Another object of the invention is to provide methods of screening test compounds useful for treating cancers.
Another object of the invention is to provide a cell useful for screening test compounds useful for treating cancers.
It is yet another object of the invention to provide a method for visualizing a human chromosomal arm 3q.
It is still another object of the invention to provide therapeutic compositions and methods for treating neoplasia.
These and other objects of the invention are achieved by one or more of the following embodiments. In one embodiment an isolated and purified p40 protein is provided. It has an amino acid sequence which is at least 99% identical to SEQ ID NO:2. Percent identity is determined using a Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1.
According to another embodiment of the invention a p40 fusion protein is provided which comprises a first protein segment and a second protein segment fused together by means of a peptide bond. The first protein segment consists of a p40 protein as shown in SEQ ID NO:2.
According to yet another embodiment of the invention a preparation of antibodies is provided. The antibodies specifically bind to a p40 protein having an amino acid sequence as shown in SEQ ID NO:2. The antibodies do not bind to p53 as shown in SEQ ID NO:4.
In still another embodiment of the invention a cDNA molecule is provided which encodes a p40 protein having an amino acid sequence which is at least 99% identical to SEQ ID NO:2. Another aspect of the invention is a cDNA molecule which is at least 99% identical to the nucleotide sequence shown in SEQ ID NO:1. In still another aspect of the invention a nucleic acid construct is provided. The construct comprises: a promoter and a polynucleotide segment encoding a p40 protein as shown in SEQ ID NO:2. The polynucleotide segment is located downstream from the promoter. Transcription of the polynucleotide segment initiates at the promoter and the promoter is not the endogenous p40 promoter. Also provided is a host cell which comprises such a nucleic acid construct.
According to another embodiment of the invention a method is provided for diagnosing and classifying a neoplastic tissue of a human. Amplification of a p40 gene is detected in a tissue suspected of being neoplastic. The p40 gene has the coding sequence shown in SEQ ID NO:1. Amplification indicates neoplasia of the tissue.
Also provided is another method of identifying or classifying a neoplastic tissue of a human. Expression of a first p40 gene in a first tissue of a human suspected of being neoplastic is compared with expression of a second p40 gene in a second tissue of the human which is normal. Increased expression of the first p40 gene relative to the second p40 gene identifies the first tissue as being neoplastic and having a p40 amplification.
According to another aspect of the invention a method is provided of screening test compounds for the ability to modulate the binding of a p40 protein to a p53 protein. A test compound is contacted with a first protein comprising a p53 protein as shown in SEQ ID NO:4 and a second protein comprising a p40 protein as shown in SEQ ID NO:2. The first and second proteins bind to each other in the absence of the test compound. The amount of the first protein which is bound or unbound to the second protein or the amount of the second protein which is bound or unbound to the first protein in the presence of the test compound is determined. A test compound which modulates the amount of bound first or second protein or which modulates the amount of unbound first or second protein is a potential drug for treating cancer.
Another aspect of the invention is a method of screening test compounds for the ability to modulate the binding of a p53 protein to a p40 protein. A cell is contacted with a test compound. The cell comprises (i) a first fusion protein comprising a p40 protein as shown in SEQ ID NO:2 and either a DNA binding domain or a transcriptional activating domain; (ii) a second fusion protein comprising a p53 protein as shown in SEQ ID NO:4 and either a DNA binding domain or a transcriptional activating domain; and (iii) a reporter gene comprising a DNA sequence to which the DNA binding domain specifically binds. If the first fusion protein comprises a DNA binding domain, then the second fusion protein comprises a transcriptional activating domain, and if the first fusion protein comprises a transcriptional activating domain, then the second fusion protein comprises a DNA binding domain. The interaction of the first and second fusion proteins reconstitutes a sequence-specific transcription activating factor. The expression of the reporter gene is measured. A test compound which modulates the expression of the reporter gene is a potential anti-cancer drug.
Also provided by the present invention is a cell which comprises three recombinant DNA constructs: a first construct encodes a first polypeptide fused to a sequence-specific DNA-binding domain; a second construct encodes a second polypeptide fused to a transcriptional activation domain; and a third construct comprises a reporter gene downstream from a DNA element which is recognized by the sequence-specific DNA-binding domain. Either the first polypeptide comprises a p40 protein as shown in SEQ ID NO:2 and the second polypeptide comprises a p53 protein as shown in SEQ ID NO:4, or the first polypeptide comprises a p53 protein as shown in SEQ ID NO:4 and the second polypeptide comprises a p40 protein as shown in SEQ ID NO:2.
Another embodiment provided by the present invention is a method of visualizing a human chromosomal arm 3q. A preparation of metaphase human chromosomes is contacted with a nucleotide probe comprising at least 12 contiguous nucleotides selected from the nucleotide sequence shown in SEQ ID NO:1. A chromosome which specifically hybridizes to the nucleotide probe is detected and identified as a human chromosomal arm 3q.
According to another aspect of the invention a therapeutic composition is provided for treating neoplasia. The composition comprises a therapeutically effective amount of an antisense p40 polynucleotide and a pharmaceutically acceptable carrier.
According to another aspect of the invention a therapeutic composition is provided for treating neoplasia. The therapeutic composition comprises a therapeutically effective amount of an antibody which specifically binds to a human p40 protein and a pharmaceutically acceptable carrier.
Yet another aspect of the invention is a method of treating neoplasia. A therapeutically effective amount of a therapeutic p40 composition is administered to a patient with neoplasia, whereby the patient""s neoplasia is reduced.
The present invention thus provides the art with an important new drug target which is important in the process of cancer development and progression. The target can be used diagnostically and therapeutically as well as in the screening and testing for new pharmacologic agents.